【作者】 韩英；

【导师】 张贵学；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 虹鳟Onchorynchus mykiss(Walbaum)是重要的冷水性养殖鱼类。为提高产品的质量和群体产量,近年来养殖人工诱导三倍体不育系虹鳟已成为一种发展趋势。四倍体虹鳟作为三倍体制种的亲本,也已被成功诱导和培育。由于染色体多倍化,多倍体虹鳟的生殖机制发生了改变,甚至导致了三倍体虹鳟雌性不育。性腺是鱼类进行繁殖活动的基础,只有充分了解性腺发育和配子发生的规律,才能对鱼类的繁殖过程更好地进行调控。尽管有关三倍体虹鳟性腺发育已有一些研究,但缺乏对其整个发育过程的组织学、细胞学、生殖内分泌学等的系统研究,尤其缺乏对早期性腺发育的了解,对三倍体虹鳟卵巢败育的起始变化过程没有描述,对卵母细胞败育的起始时间也各有不同的观点,更缺乏对性腺中非生殖细胞发育变化及其作用的研究,对三倍体虹鳟的生殖机制也有各自不同的解释。有关四倍体虹鳟的性腺发育研究尚未见报道。本研究以4~35月龄的二倍体和三倍体虹鳟以及26月龄、35月龄的四倍体虹鳟为试验材料,采用比较解剖技术、光学显微技术、透射电镜技术、放射免疫和免疫组化等技术,从性腺解剖结构、组织结构、超微结构以及生殖激素含量变化等几方面,对二倍体、三倍体和四倍体虹鳟进行了比较研究,并对二倍体和三倍体虹鳟的卵黄蛋白原进行了免疫组化定位,以了解染色体多倍化对多倍体虹鳟性腺发育和配子发生的影响,探讨多倍体虹鳟的生殖机制。此外,对不同倍性虹鳟的血细胞特征进行了研究。试验结果表明:1.二倍体雌性虹鳟35月龄卵巢已接近成熟,多数样本卵巢发育至IV+++期并排卵;雄鱼26月龄基本成熟,绝大多数样本精巢进入V期(排精期)。性成熟前雌雄虹鳟性体指数GSI持续上升,产前达峰值;雌性虹鳟肝体指数HSI在卵巢发育中后期显著下降,与GSI变化呈负相关。2.三倍体卵巢形态发育异常,不能成熟。I期卵巢与二倍体没有区别,在II期(8月龄)后发育停滞,始终保持前部稍膨大而后部呈线状的形态,没有可见的卵母细胞。个别样本卵巢中有极少数正常发育的卵母细胞。GSI始终维持在较低水平,低于同期二倍体。HSI也低于同期二倍体,与GSI没有明显的相关性;三倍体精巢形态发育基本正常,与二倍体比较发育滞后,35月龄只有少数个体成熟进入V期。GSI低于二倍体。3.四倍体雌、雄虹鳟性腺均能正常发育,形态与二倍体相似,性成熟稍晚于二倍体。35月龄仅有少数个体卵巢进入IV+++期,绝大多数样本卵巢仍处于未排卵的IV++期;26月龄仅有少数样本精巢发育成熟(V期);GSI与二倍体无显著差异。4.二倍体虹鳟卵黄发生早期的卵母细胞卵黄核呈条块状,然后迁移至皮质区形成外周环,超微结构显示,卵黄核由高尔基体和线粒体组成,为内源性卵黄的来源。生长环即将消失时,卵母细胞胞质中部出现一种嗜碱性丝状环,并消失于III时相。5.三倍体虹鳟卵巢发育阻滞,经历了卵原细胞→卵黄发育前期卵母细胞→败育卵母细胞+卵原细胞簇→卵原细胞簇+类生精细胞囊→类生精细胞囊的过程。染色体三倍化对雌鱼的影响主要发生在卵巢发育早期,发育中后期卵巢有雄性化趋势。在性腺分化期,雌性三倍体虹鳟卵巢发育与同期二倍体没有区别,卵母细胞败育发生在第一次减数分裂的双线期(核仁外周晚期、卵黄发生前期),而不是其他学者报道的偶线期到粗线期。三倍体虹鳟雌性不育是综合因素所致:卵母细胞在第一次减数分裂的双线期败育是由于已配对的染色体分离紊乱;此后,卵巢中的卵原细胞不能进一步发育,是因为被卵原细胞簇壁的基质细胞阻隔,缺乏与滤泡细胞的互作而不能进一步分化。游离于卵原细胞簇外的卵原细胞可正常发育至成熟。6.三倍体雄性虹鳟能够完成精子发生过程,较同期二倍体精巢发育迟缓、坏死细胞多、精子大且大小不等。染色体三倍化对雄鱼的影响主要发生在精子发生的晚期(精子变态期)。7.三倍体虹鳟精巢和卵巢发育中败育的生殖细胞主要被支持细胞(雄性)、类支持细胞(雌性)和成纤维细胞所吞噬。8.四倍体虹鳟卵巢和卵母细胞能够正常发育成熟;精子发生过程与二倍体基本相同,稍有滞后。其精子头部直径为2.82±0.33μm,体积明显大于三倍体(2.37±0.12μm)和二倍体(1.86±0.12μm)。9.二倍体雌性虹鳟未成熟期血清中GtH-Ⅰ和17β-E2持续升高,临近性成熟前GtH-Ⅰ和17β-E2下降。GtH-Ⅱ和T在发育前期含量很低,持续缓慢上升,在临近性成熟前迅速增加。表明GtH-Ⅰ通过刺激性腺产生17β-E2,促进卵母细胞的发育,而GTH-Ⅱ则促进卵子的成熟;三倍体雌性虹鳟生殖激素的变化与二倍体有明显不同,血清中GtH-Ⅰ、GtH-Ⅱ和17β-E2始终在低水平波动,没有明显变化规律。T含量在21月龄后持续上升,显著高于同期二倍体,为三倍体虹鳟卵巢发育类雄性化提供了生理学证据;四倍体雌性虹鳟血清中GtH-Ⅰ、GtH-Ⅱ、17β-E2和T含量与二倍体呈现相同的变化规律。10.二倍体雄性虹鳟未成熟期血清中GtH-Ⅰ和17β-E2持续升高,临近性成熟前GtH-Ⅰ和17β-E2下降。GtH-Ⅱ和T在发育前期含量很低,持续缓慢上升,在临近性成熟前迅速增加。11~35月龄雄性个体呈现了2个周期性变化;三倍体雄性虹鳟血清中GtH-Ⅰ、GtH-Ⅱ、17β-E2和T含量与二倍体有相同的变化规律,其变化较同期二倍体滞后,只有1个变化周期;四倍体雄性虹鳟血清中GtH-Ⅰ、GtH-Ⅱ、17β-E2和T含量与二倍体呈现相同的变化规律。11.采用饱和硫酸铵分步沉淀与凝胶色谱两步层析法,从虹鳟卵粗提液中分离、纯化的蛋白为含糖、磷和脂的卵黄脂磷蛋白,分子量为123.2kDa。其抗血清在无外源诱导物诱导的情况下,只与正常雌鱼血浆中的卵黄蛋白原Vg反应,为雌性特异性蛋白。12.免疫组化组织定位显示,二倍体雌性虹鳟卵巢发育至III~V期,肝细胞和血液中存在Vg,卵巢阳性反应滞后,IV~V期卵母细胞中出现Vg。其它生长期,二倍体雌性虹鳟肝细胞和血液Vg呈阴性反应;各发育期三倍体雌性虹鳟,肝脏、血液及性腺Vg呈阴性反应;各发育期雄性二倍体和三倍体虹鳟,肝脏、血液及性腺Vg均呈阴性反应,表明环境中不存在诱导卵黄蛋白原非正常表达的诱导因子;各发育期雌、雄二倍体和三倍体虹鳟肠组织Vg均呈阴性反应,表明Vg的形成与肠道无关。13.三倍体和四倍体虹鳟红细胞比二倍体更大、更长。三倍体和四倍体虹鳟红细胞大小分别为17.35±1.59×10.52±0.70μm和18.66±2.28×11.08±1.47μm ,均大于二倍体(13.30±1.31×8.34±0.63μm),与二倍体红细胞体积之比及核体积之比预期理论值(1.5︰1、2.0︰1)无显著差异;红细胞短径/长径比值分别为0.60、0.59,小于二倍体(0.63)。三倍体虹鳟红细胞压积明显小于二倍体,是由于三倍体虹鳟单位体积的红细胞数量比二倍体少所致;三倍体虹鳟的红细胞脆性小于二倍体;三倍体虹鳟与二倍体虹鳟的单位体积血液血红蛋白量没有显著差异。14.多倍体虹鳟血液中有一定比例的红细胞核呈哑铃型或双核型。低渗试验显示为红细胞异常分裂,表明多倍体虹鳟的外周血液中红细胞不是终端分化细胞,具有分裂能力。双核型红细胞是核先于质分裂,哑铃型红细胞核是核质同步分裂的中间过程。15.三倍体虹鳟血细胞的发育经历了原始、幼稚和成熟三个阶段。外周血液红细胞系、单核细胞系、淋巴细胞系和粒细胞系中未成熟血细胞的比例分别为13.8%±1.24%、4.14%±0.99%、5.52%±0.58%和3.96%±0.47%,明显高于造血器官的相应指数。外周血液中红细胞的异常分裂是其幼稚红细胞数量增多的主要原因。16.在头肾所产生的血细胞中红细胞比例为66.90%±3.85%,高于脾脏和肝脏的相应指数(56.90%±1.77%、47.20%±2.14%);在肝脏所产生的血细胞中单核细胞和粒细胞比例分别为21.80%±1.58%和4.92%±0.01%,高于脾脏和头肾的相应指数(17.80%±2.34%、4.87%±0.99%;6.82%±1.24%、3.11%±1.10%);在肝脏所产生的血细胞中淋巴细胞的比例为14.90%±1.29%,高于头肾和脾脏的相应指数(13.20±2.86%、12.60%±2.74%)。更多还原

【Abstract】 Rainbow trout (Onchorynchus mykiss Walbaum) was one of important cultivated cold water species. In order to improve quality of the products and output of the stock, cultivating artificial-inducing sterile triploid rainbow trout has come into practice in recent years. The tetraploid rainbow trout has already been inducted and cultivated successfully as the parent of breeding of triploid rainbow trout. Due to the polyploidisation of chromosome, the reproductive mechanism of the polyploid rainbow trout has changed and resulted in triploid female rainbow trout sterile. Since the gonad was the foundation of fishes for breeding, comprehensively understanding the regularity of gonadal development and gametogenesis can benefit controlling the process of breeding. Although there were some studies on the gonadal development of triploid rainbow trout, the systemic studies on histology, cytology, reproductive endocrinology have not finished in the whole developmental process, especially on gonadal development at the early stage. Up to now, there were no systemic reports about triploid ovaries from normal to diapause.This experiment was done with diploid and triploid rainbow trout of 4~35 months old and with tetraploid rainbow trout of 26 and 35 months old. The comparative studies on gonad of diploid, triploid and tetraploid rainbow trout were done in several aspects such as anatomic, histological ones and the changes of reproductive hormone. This study also made location of diploid and triploid female rainbow trout’s vitellogenin by immune histochemistry method. In addition, the blood cell characteristics of polyploid rainbow trout were researched. The conclusions were showed as follow:1. 35 month-old diploid female rainbow trout was near sexual maturity, the most of the ovary developed to stage IV+++ and ovulated. 26 month-old diploid male is up to maturity, the most of them developed to stage V (spermiation stage). The GSI of female and male rainbow trout before sexual mature was increasing constantly and reached the peak near the time of reproduction. The HSI of female rainbow trout was descending obviously in the midphase and anaphase of ovary development, and was negatively correlated to GSI.2. The ovaries of triploid rainbow trout developed abnormally and immaturely. The ovaries of triploid and diploid rainbow trout have no differences between stage I. After stage II (8 month old), ovaries diapause, the front parts of them were intumescentia slightly and the posterior parts linear. Some individuals have small number of normal developmental oocytes. The GSI was keeps in low level and lower than one of diploid individuals of the same stage. The HSI was lower than one of diploid rainbow trout and there were no obvious correlation compared with GSI. Triploid rainbow trout had morphological normal development spermary which developed slower than diploid spermary. Only a few individuals maturited and came into stage V at 35 month-old. The GSI was lower than that of diploid individuals.3. The gonadal morphology of tetraploid was similar to that of diploid, but the sexual maturation of teraploid was later than that of diploid. At 35 months old, a few ovaries developed to the stage IV+++, and mostly samples were still at the stage IV++. At 26 months old, a few spermaries developed to stage V. The GSI of tetraploid was not significantly different from that of diploid at same stage.4. The oocyte’s yolk nucleus of diploid rainbow trout looked like lump in the early egg yolk genesis, and transferred and formed outer ring at the cortex. It was the source of endogenous egg yolk, composing by Gaogi apparatus and mitochondria. Soon after the growing links were disappearing, a basophilla silkiness link formed in the oocyte’s cytoplasm and disappeared in stage III.5. The triploid rainbow trout’s ovary diapaused and underwent the process: ovogonium→oocyte in the earlier of yolk development→ovogonium cluster→grp-androgone cytocyst. The influence of chromosome triplication on female triploid fish mainly took place at the early ovary development. There was no difference between female triploid rainbow trout and diploid one in ovaries development at the gonad differentiated stages. Oocyte was abortion at the diplotene stage of meiosisⅠ(advanced stages of nucleolus periphery, protophase of Vitellogenesis) rather than at amphitene and pachynema of meiosisⅠin other reports. The ovaries had the tendency of masculinization at the midanaphase of gonadal development. The triploid female sterility resulted from comprehensive factor: the abortion of oocyte at the diplotene stage of meiosisⅠwas due to the disorder separation of the paired chromosome. After abortion of oocyte, oogonium can not further development in ovaries, this resulted from separating block of the matrix cell in the wall of ovogonium cluster. Duing to the separation, oogonium lost the interaction with the follicular cells and could not go on further differentiation. Oogonium which was liberated from outside of oogonium cluster could continue developing and maturate.6. The triploid male trout could finish spermatogenesis, but their spermary developed slower than the spermary development of the diploid in the same stage. More dead cells appeared, the sperm of triploid trout was large with the inequality of size. The impact of chromosome triplication on male triploid mainly happened in the advanced stage (stage of metamorphosis) of spermatogenesis.7. The abortive generative cells in the spermary and ovaries of triploid rainbow trout was phagocytized by sertoli’s cells (male), grp- sertoli’s cells (female) and fibroblast.8. Tetraploid female rainbow trout’s ovary and oocytes could develop normally and be fertile. The spermatogenesis was quite similar to one in diploid trout and slightly lagged behind. The diameter of sperm head was 2.82±0.33μm, and the volume was obviously bigger than triploid (2.37±0.12μm ) and diploid (1.86±0.12μm ).9. The levels of GtH-Ⅰand 17β-E2 in serum of diploid female rainbow trout increased constantly during immaturity, and decreased near maturity. The contents of GtH-Ⅱand T were low at the protophase of development, and increased slowly and constantly, but increased quickly near sexual maturity. It indicated that 17β-E2 was produced by GtH-Ⅰthrough the stimulation to gonad, and promoted the development of oocytes. GTH-II promoted the ovum to maturity. The changes of reproductive hormone in triploid female rainbow trout were obviously different from diploid, the levels of GtH-Ⅰ, GtH-Ⅱand 17β-E2 in the serum fluctuate within a low range, and the regularity was not obvious. There was a constant increasing in the levels of T in triploid female rainbow trout after 21 months and was higher than the diploid at the same stage, which provides a physiological evidence for the masculinization-like of ovaries in triploid rainbow trout. The changes of GtH-Ⅰ, GtH-Ⅱ, 17β-E2 and T in tetraploid female rainbow trout was as the same as diploid.10. The levels of GtH-Ⅰand 17β-E2 in immature diploid male rainbow trout increased constantly and became lower near sexual maturation. The contents of GtH-Ⅱand T was low at the protophase of development, increased slowly and constantly, and shew a quickly increase near sexual maturity. The male individuals had two periodic changes during 11-35months. The change s of GtH-Ⅰ, GtH-Ⅱ, 17β-E2 and T in male triploid rainbow trout were identical to ones in the diploid, but lagged behind diploid at the same stage, and existed only in one variation cycle. The contents of GtH-Ⅰ, GtH-Ⅱ, 17β-E2 and T in male tetraploid rainbow trout had the same change regularity with the diploid.11. Vitellogenin of rainbow trout’s ovum were extracted and purified by saturated ammonium sulfate fractional precipitation and gel chromatography. Lipovitellenin contained sugar, phosphorus and adipose with 123.2 kDa molecular weight. Its antiserum reacted only with the vitellogenin (Vg) in the normal female fish plasma without exogenous inducer, which indicated that it was a female peculiar protein.12. Immuno-histochemistry allocation showed that the hepatocyte and blood had Vg in diploid rainbow trout’s ovaries whichdeveloped to stage III~V, positive reaction of its ovaries was hysteresis and the oocyte had Vg during stage IV~V. In other developmentive periods, the Vg in diploid female rainbow trout’s hepatocyte and blood presented the negative reaction. The Vg in liver, blood and gonad of triploid female rainbow trout also shew the negative reaction, and the Vg in liver, blood and gonad of diploid and triploid male rainbow trout in every developmental stage had similar negative reaction, which indicated that there no inducing factor in surrounding to induce the unusual expression of vitellogenin. The Vg negative reaction appeared in intestine tissue of both male and female diploid and triploid rainbow trout in every developmental stage, illustrating the formation of Vg had not any relation with intestines.13. The red blood corpuscles were bigger, longer in triploid and tetraploid rainbow trout than the diploids. The size of triploid and tetraploid rainbow trout’s erythrocyte were 17.35±1.59×10.52±0.70μm and 18.66±2.28×11.08±1.47μm respectively, greater than the diploid (13.30±1.31×8.34±0.63μm ) , and they were all larger than diploid(13.30±1.31×8.34±0.63μm)and had no significant deviation from expectant theoretical value (1.5︰1, 2.0︰1) . The rate of short diameter to long diameter for erythrocyte was 0.60 and 0.59 respectively, and was smaller than diploid (0.63). Because of the number of erythrocyte per unit in triploid rainbow trout was smaller than in diploid, the volume of packed red cells was obviously smaller than diploid. Erythrocyte in triploid rainbow trout was less fragile than in diploid, there were no significant differences in levels of hemoglobin per unit volume blood between triploid and diploid rainbow trout.14. Some of red cell nucleus looks like dumbbell or dikaryon. Hypotonic test showed that the erythrocytes were not terminal differentiated cells in the peripheral blood of polyploid rainbow trout and had ability to division. The dikaryotic erythrocyte’s karyodieresis was earlier ones of nuclus than cytokinesis and the dumbbell-like red blood corpuscle was middle course of synchronized division in the karyoplasm and cytoplasm.15. The development of erythrocyte in triploid rainbow trout passed through primitive, inmature and ripe stages. The proportion of immature blood cell was13.8%±1.24%, 4.14%±0.99%, 5.52%±0.58% and 3.96%±0.47% respectively in the triploid rainbow trout’s erythrocyte series, monocyte series, lymphocyte series and granulocyte series, and was higher than the corresponding index of the hematogenic organ. The abnormal division of the erythrocyte in the peripheral blood was the main causes of inmature red blood corpuscles increase.16. The proportion of erythrocyte was 66.90%±3.85% in the blood cell from head kidney, which was higher than corresponding index from the spleen and liver (56.90%±1.77%, 47.20%±2.14% ) .The proportions of monocyte and granulocyte were 21.80%±1.58% and 4.92%±0.01% in the blood cells from liver, higher than corresponding index from the spleen and head kidney (17.80%±2.34%, 4.87%±0.99%;6.82%±1.24%, 3.11%±1.10%). The proportion of lymphocyte was 14.90%±1.29% in the blood cell from liver, higher than corresponding index from the head kidney and spleen(13.20±2.86%, 12.60%±2.74%).更多还原